JP6797891B2 - A pharmaceutical composition comprising an adenovirus vector - Google Patents

Description

The present invention relates to adenovirus vector preparations as aqueous mixtures or freeze-dried compositions, preparations thereof and methods for obtaining dry compositions.

The adenovirus vector is a therapeutic protein delivery platform that integrates the nucleic acid sequence encoding the therapeutic protein into the adenovirus genome, which leads to expression when the adenovirus particles are administered to the treatment subject. Be taken. The development of stable formulations for adenovirus vectors that allow storage at acceptable storage temperatures and significant shelf life has been a challenge in the art.

The human adenovirus vector is described by RK Evans et al. ('Development of stable Liquid Formulations for Adenovirus-Based Vaccines' Journal of Pharmaceutical Sciences (2004) Vol. 93, No. 10, 2458-2475). Stable formulations have been reported. However, in the art, there is still a need for formulations that ensure the stability of adenovirus vectors.

We have surprisingly found that adenovirus vectors can be particularly sensitive to the presence of salts such as sodium chloride. Therefore, the present invention is an aqueous mixture containing a low concentration of salt, particularly containing a concentration of sodium chloride of 50 mM or less, and a freeze-dried composition obtained by freeze-drying from the aqueous mixture for preparing a saladenovirus vector. (Hereinafter referred to as "dry composition"). The present invention also provides a method of using a freeze-dried composition in which the composition is reconstituted with a low salt aqueous solution, for example, injection water or an aqueous solution of a nonionic isotonic agent.

In addition, it has also been found that the inclusion of the amorphous sugar trehalose as a cryoprotectant has a further beneficial effect on the stability of the saladenovirus vector particles. Accordingly, the present invention provides an aqueous mixture and a dry composition comprising amorphous sugar trehalose or a combination of trehalose and another amorphous sugar as a cryoprotectant.

In a second aspect, the invention provides a method of lyophilizing an adenovirus vector composition that uses an annealing step in the frozen phase, thereby substantially enhancing the stability of the adenovirus particles during lyophilization. ..

FIG. 1 shows the freeze-dry cycle used in Example 1. FIG. 2 shows the freeze-dry cycle used in Example 2. FIG. 3 shows the dynamic light scattering (DLS) data obtained in Experiment 4: Panel I-Composition (a); Panel II-Composition (b); Panel III: Composition (c). FIG. 4 shows the picogreen® data obtained in Experiment 5: ○ -injection water (WFI), x-NaCl 30 mM, □ -NaCl 150 mM, ◇ -sucrose 9.25%, Δ-trehalose 9 .25%; at panel I-T1m4; at panel II-T2m4. FIG. 5 shows the freeze-dry cycle used in Example 6. FIG. 6 shows the picogreen® data obtained in Experiment 6: △ -control adenovirus strain, ▽ -negative control denatured adenovirus strain, obtained with samples obtained by x-freeze dry cycle I. Data, data obtained from samples obtained by ○ -freeze-dry cycle II. FIG. 7 shows the infectivity data obtained in Example 6: Δ-control adenovirus strain, ▽ -negative control denatured adenovirus strain, data obtained from samples obtained by x-freeze dry cycle I, ○ -Data obtained from samples obtained by freeze-dry cycle II. FIG. 8 shows the picogreen® data obtained in Experiment 7: Δ-control adenovirus strain, ▽ -negative control denatured adenovirus strain, lyophilization with + -annealing -10 ° C (3 hours). Data obtained using a freeze-drying cycle of −52 ° C./primary drying -30 ° C / secondary drying + 10 ° C (6H + 6H), freezing without x-annealing −52 ° C./primary drying -30 ° C / secondary Data obtained using freeze-drying cycle of secondary drying + 10 ° C (6H + 6H) procedure, ◇ -Slow freezing at 0.5 ° C / min without annealing / primary drying -30 ° C / secondary drying + 10 ° C ( Data obtained using the freeze-dry cycle of the procedure of 6H + 6H), freeze-drying with ○ -annealing -10 ° C (2 hours) -52 ° C / primary drying -30 ° C / secondary drying + 10 ° C (6H + 6H) Data obtained using the cycle. FIG. 9 shows the infectivity data of picogreen® obtained in Experiment 7: Δ-control adenovirus strain, ▽ -negative control denatured adenovirus strain, + -annealing -10 ° C (3 hours). Data obtained using the freeze-drying cycle of the procedure of freeze-52 ° C / primary drying -30 ° C / secondary drying + 10 ° C (6H + 6H), freezing without x-annealing -52 ° C / primary drying -30 ° C. / Data obtained using freeze-drying cycle of secondary drying + 10 ° C (6H + 6H) procedure, ◇ -Slow freezing at 0.5 ° C / min without annealing / Primary drying -30 ° C / Secondary drying +10 Data obtained using a freeze-dry cycle of the ° C. (6H + 6H) procedure, ○ -Annealing -10 ° C. (2 hours) with freezing-52 ° C./Primary drying-30 ° C./Secondary drying + 10 ° C. (6H + 6H) Data obtained using the freeze-dry cycle.

Detailed Description Contrary to reports in the art regarding the formulation of adenovirus vectors, we have developed stable formulations for, for example, human adenovirus vectors, such as all adenovirus vectors, eg, monkeys. We found that it could not be successfully applied to adenovirus vectors. The present invention describes herein a composition of an adenovirus vector in which the structural integrity and functionality of the adenovirus particles are well protected or maintained.

This novel formulation can be stored in liquid or in compositions dried at 4 ° C, 25 ° C or 37 ° C for up to 1 month, 3 months, 6 months, 1 year, 2 years or 3 years. And. In one embodiment, the dry composition can be stored at 4 ° C. for 3 years, 25 ° C. for 3 months or 37 ° C. for 1 month. It is understood that preservation is sufficient if at least 50%, at least 60%, at least 70%, at least 80% or at least 90% infectivity is retained relative to the infectivity of the starting material.

The mixtures, compositions and methods described herein retain at least 50%, at least 60%, at least 70%, at least 80% or at least 90% infectivity relative to the infectivity of the starting material at 37 ° C. Allows storage of the adenovirus vector at at least 1 month, or at 25 ° C. for at least 3 months or at 4 ° C. for at least 3 years.

The stability of an adenovirus vector can be determined, among other methods, by measuring the infectivity of the vector, eg, the retention of the infectivity during manipulation (eg, freeze-drying) or storage of the viral vector. .. The term "infectivity" means the ability of a susceptible host, i.e., a vector that invades a cell and delivers its genetic material to host expression. Infectivity can be expressed as a "50% cell culture infectious dose" (CCID ₅₀ ), which is required to infect 50% of the cells in a given cell culture. The amount of adenovirus vector to be produced. Infectivity can be assessed by measuring the proportion of cells expressing the adenovirus transgene. For example, green fluorescent protein can be used as an infectivity marker, which determines the number of cells expressing green fluorescent protein after a 24-hour incubation with the vector. Alternatively, infectivity can be assessed by determining the number of cells expressing the adenovirus hexone capsid protein after 24-hour incubation with the vector.

Adenovirus has been widely used in gene transfer applications due to its ability to achieve highly efficient gene transfer in a variety of target tissues and its large transgene carrying capacity. The adenovirus vector used in the present invention can be derived from a range of mammalian hosts. Over 100 different serotypes of adenovirus have been isolated that infect various mammalian species. These adenovirus serotypes are categorized into 6 subspecies according to sequence homology and their hemagglutination capacity (AF; B is subdivided into B1 and B2) (Tatsis and Ertl Molecular Therapy (2004). ) 10: 616-629).

In one embodiment, the adenovirus vector of the present invention is derived from human adenovirus. Such human-derived adenoviruses are Ad1, Ad2, Ad4, Ad5, Ad6, Ad11, Ad24, Ad34, Ad35, particularly Ad5, Ad11 and Ad35. Although Ad5-based vectors have been used intensively in several gene therapy clinical trials, the use of Ad5 and other human C-group adenovirus vectors has limitations due to existing immunity in the general population due to spontaneous infection. possible. Ad5 and other members of Group C of humans tend to be in the most serologically positive serotypes. In addition, immunity to existing vectors may develop as a result of exposure to the vector during treatment. Existing or developed immunity to these types of highly serum-positive vectors may limit the effectiveness of gene therapy or vaccination attempts. Therefore, another adenovirus serotype is a crucial target in the performance of a gene delivery system that can escape the host's immune response.

Thus, in another embodiment, the adenovirus vector of the invention is derived from a non-human adenovirus (also simply referred to as saladenovirus). Many adenoviruses have been isolated from non-human monkeys such as chimpanzees, bonobos, red-tailed monkeys and gorillas, and vectors derived from these adenoviruses induce a strong immune response to the transgene encoded by these vectors. (Colloca et al. (2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol.324: 361-372; Roy et al. (2010) J. of Gene Med. 13: 17-25). A particular advantage of non-human monkey adenovirus-based vectors is the relatively low cross-neutralizing antibodies of these adenoviruses in the target population. For example, cross-reactivity of certain chimpanzee adenoviruses with existing neutralizing antibody responses is present in only 2% of the target population, compared to 35% in the case of certain candidate human adenovirus vectors. ..

In certain embodiments, the adenovirus vector is a non-human adenovirus such as saladenovirus, in particular a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAd155, Pan5, Pan6, Pan7 (also referred to as C7). Or it is derived from Pan9. Examples of such strains are described in WO 03/000283, WO 2010/086189 and GB15103575. And are also available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 and other sources. Alternatively, the adenovirus vector may be derived from a non-human monkey adenovirus isolated from bonobo, such as PanAd1, PanAd2 or PanAd3. Examples of such vectors described herein can be found, for example, in WO2005 / 071093 and WO2010 / 086189. The adenovirus vector may also be derived from an adenovirus isolated from a gorilla as described in WO2013 / 52799, WO2013 / 52811, and WO2013 / 52832.

Adenovirus consists of three major proteins, hexone (II), penton base (III) and knob fiber (IV), along with several other trace proteins VI, VIII, IX, IlIa and IVa2. It has a characteristic morphology with a hedron capsid. Hexons make up the majority of the structural components of capsids, which consist of 240 trimer hexone capsomers and 12 penton bases. The hexon has three conserved double barrels and three towers at its top, each tower containing a loop from each subunit that forms the majority of the capsid. The hexon base is adenovirus serotype and highly conserved, but the surface loops are variable (Tatsis and Ertl Molecular Therapy (2004) 10: 616-629). Penton is another adenovirus capsid protein that forms a pentameric base to which fibers attach. The trimer fiber protein has a rod-like structure with knobs protruding from the penton base at each of the 12 vertices of the capsid. The primary role of fiber proteins is to anchor viral capsids to the cell surface through the interaction of knob regions with cell receptors, which is characteristic of serotypes with different fiber flexible shafts and knob region variations. (Nicklin et al Molecular Therapy 2005 12: 384-393).

Adenovirus vectors can be used to deliver the desired RNA or protein sequence for in vivo expression, eg, heterologous sequences. The vector can contain any genetic element, including naked DNA, phage, transposons, cosmids, episopes, plasmids, or viruses. An "expression cassette" (or "minigene") is a selected heterologous gene (introduced gene) and other regulatory elements required to drive translation, transcription and / or expression of its gene product in a host cell. Means a combination with.

In general, adenovirus vectors are designed so that the expression cassette is located within a nucleic acid molecule that contains another adenovirus sequence in the original region of the selected adenovirus gene. The expression cassette can optionally be inserted into an existing gene region to disrupt the function of that region. Alternatively, the expression cassette may be inserted at the site of the partially or completely deleted adenovirus gene. For example, the expression cassette is located at the site of a mutation, insertion or deletion that makes at least one gene in the genomic region selected from the group consisting of E1A, E1B, E2A, E2B, E3 and E4 non-functional. May be good. The term "non-functional" means that a sufficient amount of gene region is removed or otherwise destroyed so that the gene region can no longer produce a functional gene expression product. Optionally, the entire gene region may be removed (preferably replaced with an expression cassette). Preferably, the E1 gene of the adenovirus is deleted and replaced with an expression cassette consisting of a selective promoter, the cDNA sequence of the target gene and a poly A signal to obtain a replication defective recombinant virus.

In one embodiment, the transgene encoded by the adenovirus vector is a sequence encoding a biologically and medically useful product such as therapeutic or immunogenic protein, RNA, enzyme, or catalytic RNA. Desired RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNA, RNA aptamers, and antisense RNA. An example of a useful RNA sequence is a sequence that abolishes expression of the target nucleic acid sequence in the animal being treated.

Thus, in one embodiment, the mixture or composition described herein is prophylactic (and thus immunogenic or evasive) of a subject, such as a mammalian or human subject, depending on the transgene encoded by the adenovirus vector. Or for use in therapeutic procedures.

The transgene can encode a polypeptide or protein used, for example, for the treatment of genetic defects, as a therapeutic agent or vaccine for cancer, for the induction of an immune response, and / or for the purpose of prophylactic vaccines. As used herein, inducing an immune response means the ability of a protein, also known as an "antigen" or "immunogen", to induce a T cell and / or humoral immune response against that protein.

Immunogens expressed by adenovirus vectors that are formulated as described herein and are useful for immunizing human or non-human animals against other pathogens include human and non-human vertebrates. Infects, for example, contains bacteria, fungi, parasitic microorganisms or multicellular parasites, or is derived from cancer cells or tumor cells. For example, the immunogen can be selected from various viral families.

In one embodiment, the immunogen is derived from a filovirus such as Ebola (Zaire, Sudan, Reston, Bundibugyo and Côte d'Ivoire) or Marburg. Such antigens can be derived from viral glycoproteins (transmembrane and / or secretory) and / or viral nucleoproteins. Examples of such vectors can be found, among other things, in WO2011 / 130627.

In another embodiment, the immunogen can be selected from respiratory viruses such as respiratory syncytial virus (RSV) and other paramyxoviruses such as human metapneumovirus, hMPV and parainfluenza virus (PIV). .. Suitable antigens for RSV useful as immunogens for immunizing humans or non-human animals can be selected from fusion proteins (F), adherent proteins (G), substrate proteins (M2) and nucleoproteins (N). .. Such vectors are described in WO2012 / 089833 and PCT / EP2016 / 063297. In one embodiment, the ChAd155-RSV construct disclosed in PCT / EP2016 / 063297 is considered with respect to the disclosed compositions and methods.

In another embodiment, the immunogen can be derived from a retrovirus, such as a lentivirus such as the human immunodeficiency virus (HIV). In such embodiments, the immunogen may be derived from an HIV-1 or HIV-2 sequence, such as Gag, Pol, Nef, Env and others. Such vectors are described, among other things, in GB15103575 and WO2008 / 107370.

Alternatively, or in addition, the transgene sequence may include a reporter sequence that produces a detectable signal upon expression. Such reporter sequences include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), Other well-known in the art in which luciferases, such as membrane binding proteins including CD2, CD4, CD8, influenza blood cell agglutinin proteins, and high affinity antibodies to them are present or can be produced by more conventional means. , And above all, DNA sequences encoding fusion proteins comprising membrane binding proteins appropriately fused with antigen tag domains derived from blood cell agglutinin or Myc. These coding sequences drive their expression when associated with regulatory elements, including enzymes, radiography, colorimetric determination, fluorescence or other spectrophotometric assays, fluorescence-activated cell selection assays, and enzyme-bound immunoadsorption assays. (ELISA), radioimmunoassay (RIA) and immunoassays including immunohistochemistry produce signals detectable by conventional means.

In addition to the transgene, the expression cassette may also include conventional regulatory elements functionally linked to the transgene in a manner that allows its transcription, translation and / or expression in cells transfected with the adenovirus vector. .. As used herein, "functionally linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences that act to control the gene of interest trans or remotely. ..

Expression control sequences include suitable transcription initiation sequences, termination sequences, promoter sequences and enhancer sequences; valid RNA processing signals such as splicing and polyadenylation (polyA) signals containing rabbit β-globin poly A; stabilizing mRNA in the cytoplasm. Includes sequences to enhance; sequences that enhance translation efficiency (eg, Kozak consensus sequences); sequences that enhance protein stability; and optionally sequences that promote the secretion of the encoded product. As another sequence, a chimeric intron can be used.

A "promoter" is a nucleotide sequence that allows the binding of RNA polymerase and directs the transcription of a gene. Generally, the promoter is located in the 5'non-coding region of the gene, near the transcription start site of the gene. Sequence elements within promoters that act to initiate transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeasts, plants, viruses, and mammals (including humans). Many expression control sequences, including endogenous, native, constitutive, inducible and / or tissue-specific promoters, are known and available in the art.

Adenovirus vectors are produced by modification of wild-type adenovirus to express a heterologous gene (transduction gene) and / or to delete or inactivate an undesired adenovirus sequence. The adenovirus vector may also have altered replication capacity. For example, the vector may be of a replication defective type or may have limited replication capacity to show reduced replication capacity in non-complementary cells compared to wild-type virus. This can be done by mutating the virus, for example, by deleting a gene involved in replication, eg, deleting the E1a, E1b, E3 or E4 gene. Such modifications are known to those of skill in the art and are known in the art, for example, Roy et al., Human Gene Therapy 15: 519-530, 2004; Colloca et al. (2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; or described by WO 03/000283.

These vectors are produced using techniques known to those of skill in the art. Such techniques include conventional cDNA cloning techniques such as those described in textbooks, the use of overlapping oligonucleotide sequences in the adenovirus genome, polymerase chain reactions, and suitable methods for providing the desired nucleotide sequences. included. A particularly preferred method is Colloca et al. (2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol.324: 361-372; Roy et al. (2010) J. of Standard homologous recombination methods such as those provided in Gene Med. 13: 17-25; and WO 2010/085984 or as described in Warming et al. Nuc. Acids Res. (2005) 33: e36. Includes genetically modified engineering methods.

The adenovirus vector can be produced in any suitable cell line capable of replicating the virus. In particular, complementary cell lines can be used that supply a factor (eg, E1) that is missing from the viral vector and thus results in its replication impaired properties. Such cell lines include, but are not limited to, HeLa (ATCC Accession No. CCL2), A549 (ATCC Accession No. CCL185), HEK293, KB (CCL17), Detroit (eg, Detroit 510, CCL72) and WI. It can be -38 (CCL75) cells. All of these cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209. ECACC no. In the European Collection of Animal Cell Cultures (ECACC) of the Center for Applied Microbiology and Research (CAMR, UK). PER. As represented by cells deposited as 96022940. Other suitable parental cell lines, such as C6 ™ cells or Her96 cells (Crucell), are also available from other sources.

A particularly suitable complementary cell line is the Procell92 cell line. The Procell92 cell line is based on a Tet repressor under the human phosphoglycerate kinase-1 (PGK) promoter and HEK293 cells expressing the adenovirus E1 gene transfected with the G418 resistance gene (Vitelli et al. PLOS One). (2013) 8 (e55435): 1-9). Procell 92. S is adapted for growth under suspension conditions and is also useful for producing adenovirus vectors expressing toxic proteins (www.okairos.com/e/inners.php?m=00084, last accessed 13 April 2015 ).

Adenovirus Delivery Methods and Dosages The mixtures or compositions described herein are immune responses to transgene products delivered by vectors after delivery to mammals, preferably humans, eg, humoral (eg, antibodies). And / or it may contain one or more recombinant vectors capable of inducing a cell-mediated (eg, cytotoxic T cell) response. Recombinant adenovirus comprises a gene encoding the desired immunogen (preferably in any of its gene deletions) and can therefore be used in vaccines. Recombinant adenovirus is important for inducing an immune response and can be used as a prophylactic or therapeutic vaccine against any pathogen for which one or more antigens that can limit the spread of the pathogen have been identified and whose cDNA is available. Can be done.

Thus, in one embodiment, the mixtures and / or compositions described herein are for inducing immunity of a subject, such as a human subject. If so, the level of immunity of the selected gene can be monitored to determine the need for booster immunization. After assessing the titer of the antibody in serum, optional booster immunization may be desired.

Optionally, the mixture or composition of the invention may be prepared to contain other ingredients, including, for example, adjuvants, stabilizers, pH regulators, preservatives and the like. Such adjuvants can be administered with the antigen-encoding priming DNA vaccine to enhance the antigen-specific immune response compared to the immune response that occurs when priming with the antigen-encoding DNA vaccine. Alternatively, such an adjuvant can be administered with a polypeptide antigen administered in a dosing regimen comprising the adenovirus vector of the invention.

In some embodiments, the mixtures or compositions described herein are by intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, transdermal, intradermal, nasal or oral administration. Administered to the subject.

If the treatment regimen involves the combined administration of one or more adenovirus vectors and / or additional components, they may be dispensed together (ie, in the same mixture or composition) or in different compositions. You may. When dispensed separately, they are conveniently administered at the same location or at the same site. For example, these components may be on the same side or limb (“co-lateral” administration) or on the opposite side (eg, via a route of administration selected from intramuscular, transdermal, intradermal, and subcutaneous). Alternatively, it can be administered to the extremities (“contra-lateral” administration).

The dose of viral vector depends primarily on factors such as the condition being treated, the patient's age, weight and health, and can therefore vary from patient to patient. For example, therapeutically effective adult or veterinary doses of this viral vector are generally 1 x 10 ⁵ to 1 x 10 ¹⁵ virus particles, eg, 1 x 10 ⁸ to 1 x 10 ¹² (eg, 1 x 10). ⁸ , 5 × 10 ⁸ , 1 × 10 ⁹ , 5 × 10 ⁹ , 1 × 10 ¹⁰ , 2.5 × 10 ¹⁰ , 5 × 10 ¹⁰ , 1 × 10 ¹¹ , 5 × 10 ¹¹ , 1 × 10 ¹² particles) Contains. Alternatively, the viral vector is generally 1 × 10 ⁵ to 1 × 10 ¹⁰ plaque forming units (PFU), eg, 1 × 10 ⁵ PFU, 5 × 10 ⁵ PFU, 1 × 10 ⁶ PFU, 5 × 10 ⁶ PFU, 1 It can also be administered at a dose of × 10 ⁷ PFU, 5 × 10 ⁷ PFU, 1 × 10 ⁸ PFU, 5 × 10 ⁸ PFU, 1 × 10 ⁹ PFU, 5 × 10 ⁹ PFU, or 1 × 10 ¹⁰ PFU. The dose depends on the size of the animal and the route of administration. For example, a suitable human or veterinary dose for intramuscular injection (for an animal weighing about 80 kg) ranges from about 1 × 10 ⁹ to about 5 × 10 ¹² particles / mL per part. In some cases, a plurality of administration sites may be used. In another example, a suitable human or veterinary dose may range from about 1x10 ¹¹ to about 1x10 ¹⁵ particles for oral formulations.

For adenovirus vectors, for example, by quantitative PCR analysis (Q-PCR) using primers and probes designed on the CMV promoter region, a dilution system of plasmid DNA containing the vector genome together with the expression cassette containing the HCMV promoter is standardized. It can be quantified by using it as a curve. The copy number in the test sample can be determined by the parallel line test method. Another method of quantification of vector particles can be analytical HPLC or spectrophotometry based on A ₂₆₀ nm.

An immunologically effective amount of nucleic acid can preferably be 1 ng-100 mg. For example, a suitable amount can be 1 μg to 100 mg. Appropriate amounts of a particular nucleic acid (eg, vector) can be readily determined by one of ordinary skill in the art. An exemplary effective amount of a nucleic acid component is 1 ng to 100 μg, eg, 1 ng to 1 μg (eg, 100 ng to 1 μg), or 1 μg to 100 μg, eg, 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or It can be 1 μg. Effective amounts of nucleic acid may also include 1 μg to 500 μg, such as 1 μg to 200 μg, such as 10 to 100 μg, such as 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 75 μg, 100 μg, 150 μg, or 200 μg. Alternatively, exemplary effective amounts of nucleic acid can be 100 μg to 1 mg, such as 100 μg to 500 μg, such as 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 1 mg.

Generally, the human dose is contained in a volume of 0.5 ml to 2 ml. Thus, the mixtures and / or compositions described herein are administered in volumes of, for example, 0.5, 1.0, 1.5 or 2.0 ml human doses per immunogenic component of a single agent or combination. Can be dispensed as such.

One of ordinary skill in the art can adjust these doses depending on the route of administration and the therapeutic or vaccine application in which the recombinant vector is used. Expression levels of the transgene, or circulating antibody levels for adjuvants, can be monitored to determine the frequency of dose administration.

If more than one priming and / or booster immunization step is used, this step may be administered on an hourly, daily, weekly, monthly, or yearly basis and may include a single dose. As an example, mammals may demand one or two doses containing about 10 μg to about 50 μg of plasmid in the carrier. The amount or site of delivery is preferably selected based on the attributes and conditions of the mammal.

If so, the therapeutic level of the protein encoded by the selected transgene, or the level of the immune response to it, can be monitored to determine the need for booster immunity. After assessing the CD8 + T cell response, or optionally antibody titer, in serum, optional booster immunization may be desired. Optionally, the adenovirus vector can be delivered in a single dose or in various combination schemes, eg, in combination with a treatment regimen or course containing other active ingredients, or in a prime-boost scheme.

We have found that adenovirus vectors, in dry or liquid form, can be substantially affected by the presence of salts such as sodium chloride. Thus, the present invention relates to formulations that take into account the susceptibility of adenovirus vectors to salts such as sodium chloride, ie liquid mixtures and dry compositions. In one embodiment, the saladenovirus vector is dispensed with the liquid mixture and composition described herein.

The term "salt", as used herein, is an ionic compound, as used herein, composed of a related number of cations and anions so that there is no net charge in the product resulting from the neutralization reaction of acids and bases. For example, it means sodium chloride. The component ion may be either inorganic or organic, and may be single atom or multiple atom.

Thus, according to one embodiment, the amount of salt present in the aqueous mixture, in particular the amount of NaCl, is less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or less than 7.5 mM. Is defined as. Preferably, the composition is not completely salt-free or sodium chloride-free. Thus, according to one embodiment of the invention, salts, especially sodium chloride, are present in amounts of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, or at least 4 mM. Alternatively, sodium chloride is present in an amount of 1-50 mM, 2.5-25 mM, 2.5-15 mM, 2.5-10 mM, or 2.5-7.5 mM. According to certain embodiments, sodium chloride is present in an amount of about 5 mM.

For the purposes of defining a range, the term "between" is considered to include the end point of that range as used herein. For example, when sodium chloride is said to be present in an amount of 2.5-10 mM, a formulation in which NaCl is present at a concentration of 2.5 mM or 10 mM is included.

According to a further embodiment, the salt content of the aqueous solution for reconstitution of the dry composition, such as sodium chloride content, is also defined. According to one embodiment, the amount of salt present in the aqueous solution for reconstruction, eg, sodium chloride, is less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or less than 7.5 mM. Is.

The aqueous solution for reconstitution of the lyophilized composition may be essentially salt-free, eg, essentially sodium chloride-free. Essentially free means that the concentration of salt or sodium chloride is 0 mM or very close to it.

In a further embodiment, the aqueous solution for reconstitution of the composition is not completely free of salts or sodium chloride. Thus, salts such as sodium chloride may be present in an aqueous solution for reconstitution of the dry composition in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, or at least 4 mM. Alternatively, salts such as sodium chloride can be 1-50 mM, 2.5-25 mM, 2.5-15 mM, 2.5-10 mM or 2.5-7.5 mM in an aqueous solution to reconstitute the composition. Exists in the amount of. According to certain embodiments, salts such as sodium chloride are present in an aqueous solution for reconstitution of the composition in an amount of 5 mM.

Accordingly, the present invention also comprises a method of using the dry composition described herein, wherein the dry composition is reconstituted with an aqueous solution for reconstitution of the composition, as defined herein. provide.

The term "freeze protectant" means an excipient of a species that prevents freezing damage to what is frozen, in this case an adenovirus vector.

Suitable cryoprotectants for use in the present invention are sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltose, isomaltulose, lactulose, maltose, lactose, isomaltose, martitol, Amorphous sugars such as those selected from palatinit, sucrose, maltose, dextran, or combinations thereof. In one embodiment, the cryoprotectant is an amorphous sugar selected from sucrose, trehalose, lactose, raffinose, dextran, mannitol and combinations thereof.

In certain embodiments, the cryoprotectant or amorphous sugar is a combination of trehalose, sucrose or trehalose with a second amorphous sugar, such as one selected from sucrose, lactose, raffinose, dextran and mannitol. .. Alternatively, the cryoprotectant is trehalose, sucrose or a combination of sucrose and trehalose. In another embodiment, the cryoprotectant is trehalose or a combination of trehalose and sucrose. In yet another embodiment, the cryoprotectant is trehalose.

The cryoprotectant selected according to embodiments herein may be present in a defined amount. In certain embodiments, the aqueous mixture is at least 2.5% (w / v), at least 3% (w / v), at least 3.5% (w / v), at least 4% (w / v), at least. It contains 4.5% (w / v), at least 5% (w / v), or at least 6% (w / v) of the cryoprotectant selected above herein. In another embodiment, the cryoprotectant is less than 17.5% (w / v) in the aqueous mixture, eg, less than 15% (w / v), less than 12.5% (w / v), 11%. It is present in total amounts less than (w / v), less than 10% (w / v), or less than 9.5% (w / v). In other words, the cryoprotectant is at least 4%, at least 4.5% or at least 5% (w / v%) less than 15%, less than 12.5%, less than 11% or 10% (w / v%) in the aqueous mixture. It is present in total amounts less than v%).

The total concentration of cryoprotectant in the aqueous mixture is preferably in the range of 5-10% (w / v). In one embodiment, at least 5%, 5-15%, or 5-10% (w / v%) of trehalose is used. In one embodiment, 8%, 8.5%, 9% or 9.25% trehalose is used. In certain embodiments, the aqueous mixture comprises at least 5% (w / v%) or 5-10% (w / v%) sucrose, trehalose or a combination thereof. In another particular embodiment, the aqueous mixture comprises at least 5% (w / v) of trehalose, optionally further comprising sucrose, lactose, raffinose, dextran and mannitol.

Aqueous mixtures or dry compositions include poloxamer surfactants (eg, poloxamer 188), polysolvate surfactants (eg, polysolvate 80 and / or polysorbate 20), octoxinal surfactants, polydocanol surfactants, It may further include a polyoxyl stearate surfactant, a polyoxyl oxamer oil surfactant, an N-octyl-glucoside surfactant, a macrogol 15 hydroxy stearate, and a surfactant selected from combinations thereof. In certain embodiments, the surfactant is selected from a poloxamer surfactant (eg, poloxamer 188), a polysorbate surfactant (eg, polysorbate 80 and / or polysorbate 20), in particular a polysorbate surfactant, eg, polysorbate 80. Will be done.

In one embodiment, the surfactant is at least 0.001%, at least 0.005%, at least 0.01% (w / v), and / or up to 0.5% (w) when calculated for the aqueous mixture. It exists in the amount of / v). The surfactant may be present in an amount of less than 0.25% or less than 0.1% (w / v). In another embodiment, the surfactant is present in an amount of 0.02% (w / v).

According to certain embodiments, the surfactant is a polysorbate present in the aqueous mixture in an amount of 0.005% to 0.5% (w / v), eg, about 0.02% (w / v). 80 or poloxamer.

In a further embodiment, the buffer is added to the aqueous mixture or dry composition. The pH is generally adjusted in the light of the therapeutic component of the composition. Preferably, the pH of the aqueous mixture is at least 6, at least 6.5, at least 7 or at least 7.5. In other words, the pH of the aqueous mixture can be less than 10, less than 9.5, less than 9 or less than 8.5. In other embodiments, the pH of the aqueous mixture is 6-10, 7-9.5, 7.5-9.5, or about 7.5, such as 7.5 +/- 0.5, or 8. It is 5 +/- 0.5. Optimal pH is also determined, in part, by the particular adenovirus vector dispensed and / or the transgene integrated therein.

Suitable buffers are tris, succinate, borate, Tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate. It can be selected from salts (carbonate) or combinations thereof. In one embodiment, the buffer is tris, succinate or borate. In a further embodiment, the buffer is Tris.

The buffer can be present in the aqueous mixture in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM or at least 5 mM. Alternatively, the buffer may be present in the aqueous mixture in an amount of less than 50 mM, less than 40 mM, less than 30 mM or less than 20 mM. For example, the buffer can be present in an amount of 0.5 mM-50 mM, 1 mM-50 mM or 2 mM-20 mM. In one embodiment, the buffer is present in an amount of about 10 mM.

According to certain embodiments, the buffer is Tris present in the aqueous mixture at 2-20 mM, eg, about 10 mM.

In certain embodiments, the composition also comprises histidine in a maximum or amount of about 20 mM, eg, a concentration of about 10 mM.

According to a further embodiment, the composition also comprises divalent metal ions such as Mg ²⁺ , Ca ²⁺ or Mg ^{2+ in} the form of salts such as MgCl ₂ , CaCl ₂ or sulfonyl ₄ . In one embodiment, the divalent metal ion is Mg ²⁺ . Typical amounts of divalent metal ions present in the aqueous mixture are 0.5-10 mM, such as 1 or 2 mM, or especially 1 mM.

Excipients considered for inclusion in the composition (ie, salts, sodium chloride, cryoprotectants, buffers, surfactants and others described herein) for the purposes of describing embodiments of the invention. A particular amount of (things) is generally expressed (unless otherwise specified) as w / v% calculated relative to the volume of the aqueous mixture. Alternatively, if the aqueous mixture is freeze-dried and reconstituted, the amount of excipient may be expressed as w / v% calculated relative to the volume of the reconstituted composition.

In one embodiment, the aqueous mixture and / or (freeze-dried) composition described herein can be administered to a mammalian, eg, human subject. For dispensing as an aqueous mixture or freeze-dried composition described herein, it particularly comprises an adenovirus vector (ie, a recombinant adenovirus vector) encoding a transgene, which is a therapeutic or immunogenic protein. A mixture or composition consisting of is considered.

The aqueous mixture or dry composition can be encapsulated in either siliconized or non-siliconized glass vials. In one embodiment, the aqueous mixture or dry composition is prepared in a non-silicon treated vial. Suitable aqueous mixtures can be encapsulated in non-silicon treated vials and can be freeze-dried when encapsulated in the vials.

The present invention also provides a method for freeze-drying a liquid containing an adenovirus vector, eg, an aqueous mixture as defined herein, comprising an annealing step to obtain a dry composition. The lyophilization or freeze-drying cycle usually consists of three process phases. In the first phase of the process, most aqueous solutions or mixtures are frozen. Next, first, water is removed by sublimation during primary drying. In the third phase, non-frozen water is removed by diffusion and desorption during secondary drying. We have now found that the introduction of an annealing step during the frozen phase of the lyophilization cycle has an unexpected positive effect on the stability of the adenovirus vector.

Accordingly, the present invention also provides a method for freeze-drying a liquid containing an adenovirus vector, eg, an aqueous mixture described herein, wherein the freeze-drying cycle freezing step comprises an annealing step.

For the purposes of defining the described method, the following terms are used as is known in the art. The term "glass transition temperature" or "Tg" is the temperature at which an amorphous solid softens when heated or becomes brittle when cooled. The term "Tg'" means the frozen glass transition temperature. The term "collapse temperature" or "Tc" means the temperature at which an amorphous material softens to the extent that it can no longer support its inherent structure. The terms "freeze-dried" and "lyophilized", as well as "lyophilized" and "lyophilized" are used interchangeably to explode freeze the wet material and then dehydrate under reduced pressure, the same. Means process.

As used herein, the term "annealing step" means a method step in a freeze-drying cycle of a composition in which the product is maintained at a particular sub-zero temperature for a predetermined period of time during the freezing phase. As is known to those of skill in the art, annealing results in Ostwald ripening of ice crystals and cold concentration of amorphous matrix. Generally, the annealing temperature is (slightly) higher than Tg'. In one embodiment, the annealing is performed at a temperature of (Tg'+ 0.5 ° C.) to (Tg'+ 20 ° C.), such as -15 ° C. +/- 9 ° C. or -15 ° C. +/- 6 ° C., or ( The temperature is from Tg'+ 0.5 ° C. to (Tg'+ 10 ° C.). In any case, the annealing temperature should be from Tg'to melting temperature (Tm) during annealing. In certain embodiments, annealing is performed at temperatures of -4 ° C to -24 ° C, or -4 ° C to -20 ° C, or -4 ° C to -15 ° C, or also -8 ° C to -15 ° C. Annealing can be performed during freezing of the product, i.e., as long as the product is frozen (solid state) and in a glassy state (less than Tg'), while the frozen sample is being formed. Alternatively, annealing is performed after freezing the product.

In certain embodiments, more specifically when the aqueous mixture comprises about or at least 9% (w / v) trehalose, the annealing temperature is about −10 ° C. (eg, −10 ° C. +/- 1 ° C.). Is.

In certain embodiments, the product is frozen prior to the annealing step (ie, product temperature below Tg'). In certain embodiments, freezing is achieved by exposing the sample or aqueous mixture to a constant shelf temperature with a freezing temperature below Tg'. In another embodiment, the product can be frozen by applying shelf-ramp freezing, i.e., by gradually lowering the shelf temperature to a freezing temperature below Tg'. According to the embodiment, the freezing temperature is 5 ° C. lower than Tg', 7.5 ° C. lower than Tg', or 10 ° C. lower than Tg', for example −50 ° C. or less. According to one embodiment, the product temperature at the start of the freeze-drying cycle (ie, the temperature of the sample in the freeze-drying machine) is + 2 ° C to + 8 ° C.

When applying shelf-ramp freezing, the temperature should be at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min. And / or descent at a rate of less than 10 ° C / min, 7.5 ° C / min, 5 ° C / min or less than 3 ° C / min. Alternatively, the temperature is lowered at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min. According to a further embodiment, the achieved shelf temperature is maintained for about or at least 1 hour (or 60 minutes).

In a further embodiment for situations where the product is frozen prior to application of the annealing step, the shelf temperature is higher than Tg', eg, 0 above Tg', to initiate the annealing step after initial freezing of the sample or product. .5 ° C higher, 1 ° C higher than Tg', 3 ° C higher than Tg', 5 ° C higher than Tg', 10 ° C higher than Tg'or 20 ° C higher than Tg'. In any case, the temperature is kept below Tm during annealing. In certain embodiments, the temperature is at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min, and / or less than 10 ° C / min. , 7.5 ° C / min, 5 ° C / min or less than 3 ° C / min. Alternatively, the temperature is raised at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min. According to a further embodiment, the annealing temperature is maintained for at least 2 hours and / or up to 4 hours.

In a further embodiment, after the annealing step, the shelf temperature is below Tg', eg, 5 ° C. below Tg', 7.5 ° C. below Tg', or Tg', before starting drying under reduced pressure. The temperature is lowered by 10 ° C. below, for example, below -50 ° C. In certain embodiments, to achieve this, the temperature is at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min, and. / Or descend at a rate of less than 10 ° C / min, less than 7.5 ° C / min, less than 5 ° C / min, or less than 3 ° C / min. Alternatively, the temperature is lowered at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min. According to a further embodiment, the achieved shelf temperature is maintained for about or at least 1 hour (or 60 minutes).

Steps of the freeze-drying method described herein b. ii. The drying under reduced pressure as intended in is generally performed in two phases, namely primary drying and secondary drying. In certain embodiments, step b. ii. Is
-Steps for primary drying at a temperature lower than Tc of the product b. ii. 1. 1. Steps for secondary drying at a temperature higher than the Tc of the product and lower than the Tg of the product b. ii. 2. 2.
including.

In a further embodiment, the primary drying is performed at a pressure lower than 90 μbar and / or higher than 40 μbar. The primary drying conditions can be applied for up to 24 hours or longer.

Another embodiment is a rate of 0.1 ° C./min, at least 0.2 ° C./min, at least 0.3 ° C./min or at least 0.5 ° C./min, and / or less than 3 ° C./min, 2 ° C. With respect to the secondary drying temperature achieved by increasing the shelf temperature at a rate of less than / min or less than 1 ° C./min. Alternatively, the secondary drying temperature is achieved by increasing the shelf temperature at a rate of 0.1 to 3 ° C./min, 0.2 to 2 ° C./min, or 0.3 to 1 ° C./min. According to yet another embodiment, the secondary drying temperature is at least below −10 ° C. and / or 25 ° C. Secondary drying conditions can be applied for at least or about 3 hours, at least 4 hours, at least 5 hours, or at least or about 6 hours.

Hereinafter, the present invention will be further described with reference to the following non-limiting examples.

Example 1
The purpose of this experiment was to evaluate the effect of the annealing step during the freeze-drying cycle on the titer of ChAd3 expressing green fluorescent protein (GFP). ChAd3 particles further comprising the excipient Tris 10 mM (pH 7.4) -histidine 10 mM-MgCl 2.6H2O 1 mM-Teen 80 0.02% (m / V) -25 mM NaCl-8% sucrose (m / V). Was dispensed into an aqueous mixture of The concentration of virus particles was 2.5.10 ¹⁰ bp / ml. After the dispensing step, a 3 ml non-silicon treated type 1 glass vial was filled with a 0.5 ml ± 0.05 aqueous mixture. These vials were then partially sealed with a Helvoet FM460 bromobutyl stopper inserted in the freeze-dried position (partially inserted to allow water vapor to escape during the freeze-dry cycle). Half of the sample was transferred to the chamber of a freeze-dryer, including an annealing step (as shown in FIG. 2), and a freeze-drying cycle consisting of the following steps was performed.

1. 1. frozen:
-The shelf temperature was set to -52 ° C. When the shelf temperature fell below −45 ° C., the filled vials were loaded into the freeze-dryer. The sample was then cooled at −52 ° C. for at least 1 hour.

2. 2. Annealing process:
(1) The shelf temperature was raised to reach the target annealing temperature (-15 ° C.) in 1 hour.
(2) The annealing temperature was maintained for 2 hours.
(3) The shelf temperature was lowered again from -15 ° C to -50 ° C over 1 hour.
(4) The product was maintained at −50 ° C. for at least 1 hour.

3. 3. Primary drying:
-The chamber pressure was set to 80 μbar and the shelf temperature was raised from −52 ° C. to −30 ° C. over 3 hours. Shelf temperature and chamber pressure were maintained for 24 hours.

4. Secondary drying:
The shelf temperature was raised from −30 ° C. to 17 ° C. over 6 hours and the chamber pressure was lowered to 40 μbar. These conditions were maintained for 6 hours when the shelf temperature reached 17 ° C.

At the end of the freeze-dry cycle, the chamber was filled with dry nitrogen until the chamber pressure reached 825 mbar, and then the stopper was completely inserted into the vial (stopper ring). When the stoppering was complete, the chamber pressure was equilibrated with atmospheric pressure for removal. The chamber temperature was maintained at + 2 to + 8 ° C. until the vial was removed. These vials were then removed and oversealed with an aluminum flip-off cap.

In order to evaluate the effect of the annealing process, the freeze-dry cycle process 2. (3) and process 2. During (4), the second half of the filling vial was loaded into the chamber of the freeze-dryer.
The results of this experiment are shown in the table below.

Quantitative PCR (qPCR) reported herein makes it possible to determine virus content. This assay targets the hCMV promoter present in adenovirus. DNA samples were extracted using Qiagen QIAmp 96 DNA blood.

The Picogreen® assay measures the degradation of viral particles. The Quant-iT ™ Picogreen® dsDNA Reagent is an ultrasensitive fluorescent nucleic acid dye for quantifying double-stranded DNA in solution.

Infectivity is determined based on the amount of transgene expressed (GFP in this example). The assay measures cells expressing GFP using flow cytometric detection after 24 hours of infection.

Example 2
The purpose of this experiment was to evaluate the effect of the annealing step during the freeze-drying cycle on the titer of ChAd3 expressing eGFP. ChAd3 particles were added to the excipient Tris 10 mM (pH 7.4) -histidine 10 mM-MgCl ₂ . 6H ₂ O 1 mM-Teen 80 0.02% (w / v) -25 mM NaCl-8% sucrose (w / v) was dispensed into an aqueous mixture further comprising. The concentration of virus particles was 2.5.10 ¹⁰ bp / ml. After the dispensing step, a 3 ml non-silicon treated type 1 glass vial was filled with a 0.5 ml ± 0.05 aqueous mixture. These vials were then partially sealed with a Helvoet FM460 bromobutyl stopper inserted in the freeze-dried position (partially inserted to allow water vapor to escape during the freeze-dry cycle). Half of the sample was transferred to the chamber of a freeze-dryer, including an annealing step (as shown in FIG. 2), and a freeze-drying cycle consisting of the following steps was performed.

1. 1. frozen:
-The shelf temperature was set to -52 ° C. When the shelf temperature fell below −45 ° C., the filled vials were loaded into the freeze-dryer. The sample was then cooled at −52 ° C. for at least 1 hour.

2. 2. Annealing process:
(1) The shelf temperature was raised to reach the target annealing temperature (-15 ° C.) in 1 hour.
(2) The annealing temperature was maintained for 2 hours.
(3) The shelf temperature was lowered again from -15 ° C to -50 ° C over 1 hour.
(4) The product was maintained at −50 ° C. for at least 1 hour.

3. 3. Primary drying:
-The chamber pressure was set to 80 μbar and the shelf temperature was raised from −52 ° C. to −30 ° C. over 3 hours. Shelf temperature and chamber pressure were maintained for 24 hours.

4. Secondary drying:
The shelf temperature was raised from −30 ° C. to 17 ° C. over 6 hours and the chamber pressure was lowered to 40 μbar. These conditions were maintained for 3 hours when the shelf temperature reached 17 ° C.

At the end of the freeze-dry cycle, the chamber was filled with dry nitrogen until the chamber pressure reached 825 mbar. A stopper was attached to the vial, and when the stopper ring was completed, the chamber pressure was equilibrated with atmospheric pressure for removal. The chamber temperature was maintained at + 2 to + 8 ° C. until the vial was removed. These vials were then removed and oversealed with an aluminum flip-off cap.

To compare annealed and non-annealed samples using the same freeze-dry cycle, step 2. (3) and process 2. During (4), the second half of the filling vial was loaded into the chamber of the freeze-dryer.
The results of this experiment are shown in the table below.

qPCR, infectivity and picogreen® measurements were as described for Example 1.

Example 3
The purpose of this experiment was to evaluate the stability of the adenovirus vector when dispensed in the presence of different amounts of NaCl in the range 0-50 mM. Isotonicity was maintained by the complementary addition of sucrose. In the ChAd3Eboz construct, the adenovirus vector, chimpanzee adenovirus 3, used in this experiment is used as a vector encoding the Zaire strain Ebola glycoprotein (as described in WO2011 / 130627). Thus, the conditions listed in Table 1, were tested using 5,0.10 ⁹ vp / ml dose of ChAd3Eboz.

Samples are maintained at 30 ° C. for 3 days, after which the effect on stability of adenovirus particles is measured by qPCR (measure virus content by targeting promoter sequences) and infectivity (adenovirus hexone 24 hours after infection) The infectivity of adenovirus particles was measured by detecting the flow cytometry of stained cells for capsid protein). The test results are listed in Table 1.

qPCR, infectivity and picogreen® measurements were as described for Example 1.

Example 4
In this example, three compositions (compositions (a), (b) and (c)) were evaluated by dynamic light scattering (DLS) and each was subject to three different storage conditions for freeze-dried products. .. The storage conditions tested were 1 month at 4 ° C. (T1m4), 1 week at 25 ° C. (T1w25) and 3 days at 37 ° C. (T3d37). The freeze-dry cycle applied is the same as in Example 1 (Fig. 1).

Composition ^{(a): ChAd3Eboz 1.10 11 vp} / ml, Tris 10 mM pH 7.5, histidine 10 mM, NaCl 25 mM, sucrose 8%, MgCl ₂ 1mM, polysorbate 80 0.02%
Composition ^{(b): ChAd3Eboz 1.10 11 vp} / ml, Tris 10 mM pH 7.5, histidine 10 mM, NaCl 6 mM (residual), 7% trehalose, sucrose 2% (residual), MgCl ₂ 1 mM, Polysorbate 80 0.02 %
Composition ^{(c): ChAd3Eboz 1.10 11 vp} / ml, Tris 10 mM pH 7.5, histidine 10 mM, NaCl 6 mM (residual), 7% trehalose, sucrose 2% (residual), MgCl ₂ 1 mM, Poloxamer 188 0.15 %

Two samples of ChAd3Eboz starting material before and after treatment at 60 ° C. for 30 minutes were used as positive and negative controls, respectively.
The results of the experiment are shown in FIG.

Example 5
The ChAd3Eboz, (A) Sucrose 8% NaCl 25 mM, Tris 10 mM pH 7.4, histidine 10 mM, MgCl ₂ 1 mM and polysorbate 80 0.02%, or (B) 7% trehalose, sucrose 2% (residual), NaCl 6 mM , Tris 10 mM pH 7.4, histidine 10 mM, MgCl 2 1 mM and polysorbate 80 0.02%. A freeze-dried cycle without an annealing step was applied as described for Example 1 to obtain freeze-dried samples. The following rehydration media were tested:
-After storage of the dry composition at 4 ° C. for 1 month (T1 m4): NaCl 150 mM, NaCl 30 mM, and water injection-After storage of the dry composition at 4 ° C. for 2 months (T2 m4): NaCl 150 mM, sucrose 9 .25%, trehalose 9.25% and water injection

Two samples of both bulk ChAd3Eboz before and after treatment at 60 ° C. for 30 minutes were used as positive and negative controls, respectively.

Capsid disruption during reconstruction of the dry composition was evaluated using the Picogreen® assay. Quant-iT ™ Picogreen® is an ultrasensitive fluorescent nucleic acid for quantifying double-stranded DNA in solution.

Figure 4. As shown in the graph of I (at T1 m4 ° C.), the free DNA in the external phase was directly proportional to the NaCl concentration of the rehydration medium (WFI <NaCl 30 mM <NaCl 150 mM). In addition, the trehalose-based formulation (A) provided better capsid stability than the sucrose-based formulation (B) when using low-salt rehydration media (WFI and NaCl 30 mM).

Figure 4. As shown in the graph of II (at T2m4 ° C.), salt-free rehydration media (sucrose 9.25% w / v and trehalose 9.25% w / v) were used in low temperature samples stored at 4 ° C. for 2 months. ) Was used, the results were comparable to WFI. Also in this case, the preparation (A) based on trehalose is sucrose when a low salt concentration rehydration medium (WFI, trehalose 9.25% w / v and sucrose 9.25% w / v) is used. It gave better capsid stability than the formulation (B) based on.

Example 6
The purpose of this experiment was to evaluate the feasibility of freeze-drying the ChAd155 vector under the same conditions as described above for the ChAd3 vector. The ChAd155 vector used in this experiment encodes a respiratory syncytial viral protein and is described in PCT / EP2016 / 063297.

The conditions evaluated were as follows.
-The freeze-drying cycle applied in Example 1 (see FIG. 1, hereinafter referred to as cycle I) was subjected to an annealing step at −10 ° C. and secondary drying only by raising to 10 ° C. over 6 hours, and stopped at this point. Compare with the freeze-dry cycle (see FIG. 5, hereinafter referred to as cycle II) including the same procedure as in FIG. 1 except for the above.
The effects of trehalose and histidine contents on the four compositions:
Composition (a): ChAd155 1.10 ¹¹ pU / ml, Tris 10 mM pH 8.5, Polysorbate 80 0.02%, MgCl ₂ 1 mM, Trehalose 9%, NaCl 8 mM, Sucrose 2.5%, Histidine 10 mM
Composition (b): ChAd155 1.10 ¹¹ pU / ml, Tris 10 mM pH 8.5, Polysorbate 80 0.02%, MgCl ₂ 1 mM, Trehalose 9%, NaCl 8 mM, Sucrose 2.5%
Composition (c): ChAd155 1.10 ¹¹ pU / ml, Tris 10 mM pH 8.5, Polysorbate 80 0.02%, MgCl ₂ 1 mM, Trehalose 7%, NaCl 6 mM (residual), Sucrose 2.5%, Histidine 10 mM
Composition (d): ChAd155 1.10 ¹¹ pU / ml, Tris 10 mM pH 8.5, Polysorbate 80 0.02%, MgCl ₂ 1 mM, Trehalose 7%, NaCl 6 mM (residual), Sucrose 2.5%
Was evaluated by comparing.

Reconstitute the freeze-dried product with water injection.

Two samples of both bulk ChAd155 before and after treatment at 60 ° C. for 30 minutes were used as positive and negative controls, respectively.

Destruction of capsids during reconstruction of dry compositions, Quant-iT ™ picogreen® assay (ultra-sensitive fluorescent nucleic acid for quantifying double-stranded DNA in solution) and infectivity (adeno) The infectivity of adenovirus particles was measured by flow cytometry detection of cells stained with virus hexone capsid protein).
The results of this experiment are shown in the table below (see glasses in Figures 5 and 6):

Example 7
The purpose of this experiment was to confirm the protective effect of the annealing process on the integrity of the product and to measure the effect of the desorption rate processed during secondary drying.

Using the freeze-drying cycle selected in Example 6 (see FIG. 5), the desorption rate up to 12 hours was evaluated by adding the 3-hour tray annealing step and the secondary drying tray 6 hours. The evaluation was based on the composition of Example 6 (a): ChAd155 1.10 ¹¹ pU / ml, Tris 10 mM pH 8.5, Polysorbate 80 0.02%, MgCl ₂ 1 mM, Trehalose 9%, NaCl 8 mM, Sucrose 2.5. %, Based on histidine 10 mM.

The freeze-dried product was reconstituted with water injection.

Two samples of both bulk ChAd155 before and after treatment at 60 ° C. for 30 minutes were used as positive and negative controls, respectively.

Destruction of capsids during reconstruction of dry compositions, Quant-iT ™ picogreen® assay (ultrasensitive fluorescent nucleic acid for quantifying double-stranded DNA in solution) and CCID50 (infected host) 50% of the cells were killed or inoculated using the infectivity of the virus required to induce the cytopathic effect (CPE) in 50% of the cell cultures).

The results of this experiment are shown in the table below and are shown in FIGS. 8 and 9.

Claims (59)

Non-human monkey adenovirus vector and
Trehalose, a cryoprotectant is amorphous sugar which is selected from the scroll scan your and combinations thereof,
Surfactants selected from poloxamer surfactants and polysorbate surfactants,
With a buffer
Divalent metal ion salts selected from MgCl ₂ , CaCl ₂ and sulfonyl ₄ and
An aqueous mixture comprising an amount of sodium chloride from 0 to 50 mM. The aqueous mixture according to claim 1, which is used to store the non-human monkey adenovirus vector. Wherein sodium chloride is present in an amount of 0～40M M, aqueous mixture of claim 1 or 2. The aqueous mixture according to any one of claims 1 to 3 , wherein the sodium chloride is present in an amount of 5 mM +/- 1 mM. The aqueous mixture according to any one of claims 1 to 4 , wherein the amorphous sugar is present in an amount of at least 2.5% (w / v ) . Wherein the amorphous sugar is present in 17.5% (w / v) amount of less than, the aqueous mixture according to any one of claims 1-5. The aqueous mixture according to any one of claims 1 to 6 , wherein the amorphous sugar is present in an amount of 8%, 8.5%, 9%, or 9.25%. Comprising the trehalose at least 5% (w / v), aqueous mixture of any one of claims 1-7. The non Hitosaru adenoviral vector is selected from the volume Novo adenoviral vectors and gorillas adenoviral vector, an aqueous mixture according to any one of claims 1-8. The non Hitosaru adenoviral vector is a chimpanzee adenovirus vector over, the aqueous mixture according to any one of claims 1-8. The non Hitosaru adenoviral vector, ChAd3, ChAd63, ChAd83, ChAd155 and a luer adenoviral vectors are selected from PanAd3, the aqueous mixture according to any one of claims 1-8. The aqueous mixture according to claim 11, wherein the non-human monkey adenovirus vector is ChAd155. The surfactant, Po Rokisama 18 8, selected from polysorbate 80 and polysorbate 2 0, the aqueous mixture according to any one of claims 1 to 12. The aqueous mixture according to any one of claims 1 to 13 , wherein the surfactant is poloxamer 188 or polysorbate 80. The aqueous mixture according to any one of claims 1 to 14 , wherein the surfactant is polysorbate 80. Wherein the surfactant at least 0.001% comprising Nde containing an amount of (w / v) and up to 0.5% (w / v), aqueous mixture of any one of claims 1 to 15. Wherein the surfactant 0.02% consisting Nde containing at (w / v) the amount of the aqueous mixture according to any one of claims 1-16. The aqueous mixture according to any one of claims 1 to 17 , wherein the pH is at least 6 . pH is 10 is less than, the aqueous mixture according to any one of claims 1 to 18. The aqueous mixture according to any one of claims 1 to 19 , which has a pH of 7.5 to 9.5. The aqueous mixture according to any one of claims 1 to 20 , which has a pH of 7.5 (+/- 0.5). The aqueous mixture according to any one of claims 1 to 21, wherein the buffer is a Tris buffer in an amount of 0.5 mM to 50 mM. The aqueous mixture according to any one of claims 1 to 22 , wherein the divalent metal ion salt is present in an amount of 0.5 to 10 mM. The aqueous mixture according to claim 23, wherein the divalent metal ion salt is present in an amount selected from 1 mM and 2 mM. The aqueous mixture according to any one of claims 1 to 24 , wherein the non-human monkey adenovirus vector comprises a transgene which is an immunogenic transgene. A composition freeze-dried from the aqueous mixture according to any one of claims 1 to 25 . The composition of claim 26, which is used to store the non-human monkey adenovirus vector. A composition according to claim 26 or 27, in terms of the composition containing sodium chloride in 50mM less than the amount, or sodium chloride reconstituted with an aqueous solution not containing essentially human A composition for use in a method of administering the composition to a subject. The composition according to any one of claims 26 to 28 , wherein the composition is reconstituted with an aqueous solution containing a nonionic isotonicity adjusting agent, and then the composition is administered to a subject. Composition for use in a manner that 29. The composition of claim 29, wherein the nonionic isotonicity modifier is sucrose, trehalose or a combination thereof. The composition according to any one of claims 26 to 30 , wherein the non-human monkey adenovirus vector comprises a transgene which is an immunogenic transgene. The composition according to any one of claims 26 to 31 or any one of claims 1 to 25 for use in a method for inducing an immune response against an immunogenic transgene product in a human subject. Aqueous mixture of. a. A step of preparing an aqueous mixture comprising an adenovirus vector and an amorphous sugar selected from trehalose, sucrose and combinations thereof ,
b. The aqueous mixture is i. Freezing of the aqueous mixture, including the annealing step, and ii. A method for producing a composition comprising an adenovirus vector, which comprises a step of freeze-drying the frozen aqueous mixture by drying under reduced pressure. a. A step of preparing an aqueous mixture comprising an adenovirus vector and an amorphous sugar selected from trehalose, sucrose and combinations thereof ,
b. The aqueous mixture is i. Freezing the aqueous mixture including the following steps:
1. 1. The step of freezing the aqueous mixture by lowering the temperature below Tg'of the mixture.
2. 2. A step of applying an annealing step to the frozen mixture by raising the temperature without thawing the frozen mixture.
3. 3. The step of lowering the temperature below Tg'again, and ii. A method for producing a composition containing an adenovirus vector, which comprises a step of freeze-drying the frozen aqueous mixture by drying under reduced pressure. Step a. 33 or 34. The method of claim 33 or 34 , wherein the aqueous mixture is prepared in a non- silicon treated vial. Step a. The aqueous mixture is prepared at a temperature of +. 2 to + 8 ° C. In Method according to any one of claims 33-35. Vial comprises an adenoviral vector of a single dose, method of any one of claims 33-36. Step b. i. 1. 1. 34. The method of claim 34 , wherein the product temperature is lowered by applying a shelf temperature at least 10 ° C. lower than Tg'. Step b. i. 1. 1. 34. The method of claim 34 or 38 , wherein the product temperature is lowered by applying a shelf temperature of −50 ° C. Step b. i. 1. 1. 34. The method of any one of claims 34, 38 and 39 , wherein the temperature is lowered at a rate of 2-10 ° C./min. Step b. i. 1. 1. 34. The method of any one of claims 34 and 38-40 , wherein the temperature is maintained below Tg'for at least 1 hour. Step b. i. 2. 2. 34. The method of any one of claims 34 and 38-41 , wherein the temperature is raised to a temperature higher than Tg'of the frozen mixture and lower than the melting point of the frozen mixture. Step b. i. 2. 2. The temperature at - is raised to 15 ° C. +/- 6 ° C. annealing temperature, the method according to any one of claims 34 and 38-42. Step b. i. 2. 2. 34. The method of any one of claims 34 and 38-43 , wherein the temperature is raised to the annealing temperature at a rate of 0.1 to 10 ° C./min. Step b. i. 2. 2. 34. The method of any one of claims 34 and 38-44 , wherein the frozen mixture is maintained at the annealing temperature for 2-4 hours. Step b. i. 3. 3. 34. The method of any one of claims 34 and 38-45 , wherein the temperature is lowered below Tg'. Step b. i. 3. 3. 34. The method of any one of claims 34 and 38-46 , wherein the temperature is lowered at a rate of 0.1 to 10 ° C./min. Step b. i. 3. 3. 34. The method of any one of claims 34 and 38-47 , wherein the frozen mixture is kept below Tg'for at least 1 hour. Step b. ii. Is 1. Primary drying at a temperature lower than the decay temperature, and 2. The method according to any one of claims 33 to 48 , comprising secondary drying at a temperature higher than the decay temperature. Step b. ii. 1. 1. 49. The method of claim 49 , wherein the pressure inside is 40-90 μbar. Step b. ii. 1. 1. 49 or 50 , wherein the method is applied for at least 24 hours. Step b. ii. 2. 2. To a temperature of -10 ° C to 25 ° C , 0 . The method according to any one of claims 49 to 51 , wherein the temperature is increased at a rate of 1 to 3 ° C./min. Step b. ii. 2. 2. The method according to any one of claims 49 to 52 , wherein the temperature raised in the above is maintained for up to 6 hours. The method according to any one of claims 49 to 53 , wherein the pressure during the secondary drying is 15 to 60 μbar. Claim 1-32 further comprising a one or more of the features defined in the method according to any one of claims 33 to 54. In the aqueous mixture according to any one of claims 1 to 25 or the composition according to any one of claims 26 to 32, the aqueous mixture or the non-human monkey adenovirus contained in the composition. How to save a vector. The method of claim 56, wherein the non-human monkey adenovirus vector is stored for at least one month. The method of claim 56 or 57, wherein the non-human monkey adenovirus vector is stored at 4 ° C, 25 ° C or 37 ° C. The method according to any one of claims 56 to 58, wherein the non-human monkey adenovirus vector retains at least 50% of the infectivity of the non-human monkey adenovirus vector at the start of storage.